© Agricultural and Food Science Manuscript received January 2004
Factors infl uencing somatic embryogenesis and regeneration ability in somatic tissue culture of
spring and winter rye
Rui Ma and Seppo Pulli
Laboratory of Plant Physiology and Molecular Biology, Department of Biology, FI-20014 University of Turku, Finland, e-mail: seppo.pulli@koti.luukku.com
Rye is an important crop in Northern and Eastern Europe. However, the application of various biotechnolo- gies in rye breeding has been limited duo to its recalcitrant in tissue culture. In order to improve somatic tissue effi ciency, key factors affecting somatic embryogenesis and reproducible green plant regeneration of rye (Secale cereale L.) were evaluated and optimised. In this study, a total 27 rye genotypes including 10 spring and 17 winter genotypes were involved in the investigation. Genotype, culture medium, sugar, gel agent and auxin infl uenced somatic embryogenesis of immature embryo signifi cantly. One-two weeks cold pretreatment of young embryo enhanced somatic embryogenesis and green plant regeneration. In culture of immature embryos, infl orescences and leaf segments of the seedlings, explants signifi cantly infl uenced the culture effi ciency. Highest embryogenic callus yield resulted from rye immature embryo as explant com- pared to young infl orescence and leaf segment of seedling. Developmental stage of embryo played an im- portant role in somatic embryogenesis. Late spherical coleoptile stage (embryo size 0.5–1mm in length) was optimal developmental stage of immature embryo for culture. Morphogenetic potential of embryo- genic callus decreased with an increasing number of subcultures, and this ability could be maintained in vitro for a maximum of 8 months of culturing.
Key words: rye, somatic embryogenesis, embryo, infl orescence, leaf segment
Introduction
Various biotechnological applications in plant breeding, such as gene transformation and in vitro
selection, rely on the availability of effi cient plant tissue culture systems. Regeneration of plants from in vitro-cultured cells is an important ad- vance in the genetic manipulation of plants (Lörz et al. 1988). Plant tissue and cell culture techniques
have long been recognized as valuable tools in crop improvement programs, particularly in crop breeding. Effi cient plant tissue culture systems have been established and advances made in the genetic transformation of staple cereals, including wheat (Vasil et al. 1993, Zhang et al. 2000), rice (Dong et al. 2001, Pragya et al. 2002) and barley (Wan and Lemaux 1994, Trifonova 2001).
Rye (Secale cereale L. 2n = 2x = 14) is an im- portant cereal crop in Europe, and its adaptability to adverse conditions, such as low temperatures and diseases, among others, is also of major in- terest to plant breeders. However, rye has proved to be among the most recalcitrant graminaceous species in plant tissue culture and genetic trans- formation. So far, transgenic rye plants have been reported (Castillo et al. 1994, Popelka and Altpeter 2003a, b, Altpeter et al. 2004), and the target ma- terials were limited in specifi c inbred line. The lack of an effi cient in vitro culture system is still a limitation for genetic transformation and ma- nipulation of this species. Early investigations of rye somatic embryogenesis and plant regeneration from various explant sources of immature embryo (Rybczynski 1979, Zimny and Lörz 1989, Rakoc- zy-Trojanowska and Malepszy 1995, Popelka and Altpeter 2001), young infl orescence (Krumbiegel- Schroeren et al. 1984, Linacero and Vazquez 1990, Rakoczy-Trojanowska and Malepszy 1993), young leaf segment (Linacero and Vazquez 1986) and root organ cultures (Whitney 1996) have been described in several reports. However, the low embryogen- esis and green plant regeneration are problemati- cal. In particular, the effects of some important fac- tors, such as different explants, cold pretreatment, genotype, medium, auxins and sugars, infl uencing somatic embryogenesis remain obscure.
In this study, the important physiological and physical factors infl uencing embryogenic callus induction and reproducible green plant regenera- tion were evaluated and optimised to improve ef- fi ciency of plant tissue culture systems of rye in practical rye breeding work for genetic transfor- mation, manipulation and in vitro selection. Em- bryogenic calli from somatic tissue culture were also suitable sources for cell suspension culture initiation and protoplasts culture.
Material and methods
Plant materials
A total of 27 rye cultivars including 10 spring and 17 winter genotypes served as donor plants in this experiment (Table 1). Plant materials, which were used for immature embryo culture and in- fl orescence culture, were grown in a greenhouse (18/16ºC, day/night temperature, 16-h photoperiod at about 200 µmol m-2s-1). Seedlings of winter gen- otypes were vernalized in a cold room (4ºC, 12-h photoperiod at 70 µmol m-2s-1 light intensity) for 10–12 weeks then transferred to the greenhouse.
For leaf segment culture, sterilized seed (surface sterilized by 1.6% sodium hypochlorite with 0.5 Tween 20 for 15 min then rinsed several times with sterile water) of rye cultivars was germinated in Magenta boxes with sterilized fi lter paper at
Table 1. Source of rye materials in experiment.
Eco-type Genotype Source
Spring Auvinen Finland
ME-80083 Finland
OD Sweden
Vågones vårrug Norway KVL-7002 Denmark
Rogo Germany
Florida dwarf USA Florida 401 USA Kalhek K131 Afghanistan
Gansu China
Winter Anna Finland
Riihi Finland
Jussi Finland
Akusti Finland
Voima Finland
Elvi Estonia
Sangaste Estonia
Vambo Estonia
Vågones Norway
Amilo Poland
Danko Poland
Zulpan Russia
EM-1 Russia
Bylina Ukraina
Bonel USA
Wheeler USA
Lanzhou China
24ºC in darkness, and then grown at 24ºC/18ºC (day/night) under a light regime of 16-h photope- riod at 80 µmol m-2s-1.
Embryogenic callus induction
Spikes were harvested 10–20 days after isolated pollination (spikes isolated with cellophane bags to prevent cross pollination from other genotypes), and were stored at 4ºC in darkness with stalks in water. Immature caryopses were surface-sterilized by 1.6% sodium hypochlorite with 0.5% Tween 20 for 15 min then rinsed several times with sterile water. Immature embryos were placed on an induc- tion medium with scutellum up. Tillers containing young infl orescence 0.5–2 cm in length were col- lected, and surface-sterilized in 70% ethanol for 30 seconds. Infl orescences were cut into 2-mm-long segments and cultured on an induction medium.
For leaf segment culture, basal part of shoot (ap- prox. 30 mm, 2–3 weeks old) grown in Magenta boxes were cut into 3-mm-long sections and cul- tured on induction medium. Approximately 10 im- mature embryos, infl orescence segments or leaf sections were cultured in a Petri dish (diameter 95 mm) containing 30 ml solid induction medium.
Media MS (Murashige and Skoog 1962), CC (Potrykus et al. 1979), AA (Müller and Grafe 1978, omitted kinetin and GA3), mMS (modifi ed MS medium consisting of MS basic salts and vita- mins, 2 mg l–1 glycine, 146 mg l–1 glutamine, 200 mg l-1 casein hydrolysate, Li et al. 1992) and S1 (Yin et al. 1993) consisting of macronutrients of AA medium, micronutrients and vitamins of B5 medium (Gamborg et al. 1968), 500 mg l-1 Pro- line, 877 mg l-1 glutamine, 266 mg l-1 aspartic acid, 288 mg l-1 arginine, 75 mg l-1 glycine, 20 mg l-1 coconut milk were used as induction media. pH of the media was adjusted to 5.8 before autoclav- ing. Coconut milk was added after autoclaving by fi lter sterilization. Cultures were incubated at 27ºC in darkness. The effects of different auxins (2 mg l-1 2,4-D, Sigma, 4 mg l-1 Dicamba, Sigma, 2 mg l-1 NAA, Sigma, the concentrations used were op- timal concentrations in our pre-tests), sugars (3%
of sucrose, maltose and glucose) and gel agents
(0.3% Phytagel, Sigma and 0.7% Agar, Difco) in AA medium on embryogenesis of immature em- bryos were tested. Totally 9 050 embryos were used in these three tests.
In cold treatment test, spikes with immature embryos were cold treated at 4ºC in darkness with stalks in water for different durations (0–5 weeks).
A total of 3 600 immature embryos were used in the cold treatment experiment. Totally 1 808 im- mature embryos were used for test of genotypic effects. The culture abilities of different explants (immature embryos, infl orescence segments or leaf sections of seedling) were tested and 850 im- mature embryos, 850 immature infl orescence seg- ments and 850 leaf sections were cultured. AA me- dium was used as embryogenic induction medium in these experiments.
Effects of developmental stages of immature embryos on embryogenesis were studied. Imma- ture embryos in different developmental stages (1–
6 scale of developmental stages of Zimny and Lörz 1989, embryo size from < 0.5 mm, 0.5–1 mm, 1–2 mm, 2–3 mm) were cultured on AA medium in this experiment. Totally 3200 embryos were used in this experiment.
Green plant regeneration
After 4–5 weeks incubation, frequencies of ex- plants forming embryogenic calli were scored. To test regeneration capability, embyogenic calli were transferred onto 190-2 (Wang and Hu 1984) re- generation medium supplemented with 3% (w/v) sucrose, 3 mg l-1 BA, solidifi ed with 0.45% Phyta- gel, and cultured at 21ºC and 16-h photoperiod (40 µmol m-2s-1). Partial embryogenic calli were main- tained by monthly subculture on solid AA medium with 2 mg l-1 2,4-D, and the morphogenetic com- petencies examined at monthly intervals.
Data collection and statistical analysis
Frequencies of explants forming embryogenic calli (embryogenic calli / 100 explants) and frequencies of regenerated calli producing green plants (green
plants / 100 calli) were recorded. In the experi- ments, the completely randomized designs (CRD) were used. Each Petri dish was considered an ex- perimental unit and each treatment contained fi ve replicates. Response variables were callus induc- tion and green plant regeneration. Data analyses with more than two treatment levels were carried out by the ANOVA procedure. Multi-range com- parisons were performed by LSD test.
Results and discussion
Medium effects on somatic embryogenesis
In the fi ve media tested, AA medium produced a signifi cantly (P < 0.05) higher embryogenic ef-
fi ciency than CC and mMS media, and CC and mMS media, in turn, gave a signifi cantly (P <
0.05) higher embryogenic effi ciency than S1 and MS media. No interactions of genotype × medi- um on somatic embryogenesis were detected (P >
0.05) (Table 2).
Culture medium is another important factor infl uencing somatic embryogenesis. In previous work on the somatic tissue culture of cereal crops, different basal culture media have been employed for embryogenic callus induction. MS or modifi ed MS medium are most commonly used as induction media for somatic embryogenesis of wheat, rice and barley. CC medium has been recommended for embryo culture of rye (Zimny and Lörz 1989) and barley (Lührs and Lörz 1987). AA is an amino acid based medium preferred by Immonen (1996) for embryo culture of triticale. In this study, AA
Table 2. Effects of ecotypes, genotypes and media on somatic embryogenesis (embryogenic calli per 100 embryos) in im- mature embryo culture of rye (3% sucrose and 0.3% Phytagel were used). Each treatment contained fi ve replicates.
Eco-type Genotype Medium
AA CC mMS S1 MS
Spring ME80083 72.35 67.50 56.58 35.00 37.50
Florida dwarf 70.99 61.29 64.45 45.83 60.71
Vågones vårrug 65.53 64.62 61.11 52.34 56.67
Auvinen 63.88 57.04 53.13 44.36 39.50
OD 61.54 45.45 51.25 38.75 35.38
Gansu 61.30 32.23 44.30 39.38 30.83
Rogo 63.33 62.50 55.92 48.33 41.89
Average 65.56A 55.80B 55.25B 43.43C 43.21C
Winter Zulpan 68.14 63.04 52.94 50.00 46.67
Amilo 69.91 50.00 58.82 46.15 46.32
Jussi 63.29 56.98 53.54 43.01 37.58
Anna 66.59 62.71 63.92 54.00 49.00
Riihi 57.07 51.36 48.80 31.25 31.54
Vågones 60.61 54.33 56.25 38.55 35.05
Wheeler 58.42 52.56 50.46 37.89 43.53
Voima 54.38 49.52 48.95 38.60 37.43
Average 55.38A 48.94B 48.19B 37.72C 36.35C
Total average 63.82A 55.41B 54.69B 42.89C 41.97C LSD0.05 = 5.19 between media within spring genotypes
LSD0.05 = 5.06 between media within winter genotypes LSD0.05 = 3.62 between media
AA medium (amino acid based medium, Müller and Grafe 1978) CC medium (Potrykus et al. 1979)
mMS medium (modifi ed MS medium, Li et al. 1992) MS medium (Murashige and Skoog 1962)
S1 medium (Yin et al. 1993)
medium produced the highest number of embryo- genic calli, suggesting AA medium to be the in- duction medium of choice in practical plant tissue culture for somatic embryogenesis of rye. The rest experiments of this study were performed with AA medium (Tables 3, 4 5, 7, 8, 9, 10).
Infl uence of auxins on somatic embryogenesis
Different auxins 2,4-D, Dicamba and NAA were tested in this experiment. Signifi cantly highest em- bryogenesis was from Dicamba comparing 2,4-D and NAA. No statistically signifi cant interactions between genotype and auxin on embryogenesis were found. The highest embryogenesis frequency was from ‘Florida dwarf’ by Dicamba, the lowest was from ‘Auvinen’ with NAA (Table 3).
Effi ciency of auxins on somatic embryogen- esis in cereals has been assessed in previous works
(Lührs and Lörz 1987, Popelka and Altpeter 2001).
The type and concentration of auxins in the induc- tion medium is important for obtaining a high effi - ciency of somatic embryogenesis (Zimny and Lörz 1989). Dicamba has proved to be more effective on somatic embryogenesis than 2,4-D in embryo culture of barley (Lührs and Lörz 1987, triticale (Immonen 1996) and rye (Zimny and Lörz 1989).
In our study, Dicamba gave the best somatic em- bryogenesis compared to 2,4-D, while NAA was in accordance with the observations of Zimny and Lörz (1989).
Sugar and gelling agent in culture medium
In this study (Table 4) somatic embryogenesis was signifi cantly (P < 0.001) infl uenced by sugars used in the induction medium. Signifi cantly highest embryogenesis was produced by sucrose than by
Table 3. Effects of ecotypes, genotypes and different auxins on somatic embryogenesis (embryogenic calli per 100 em- bryos) in rye embryo culture. AA medium with 3% sucrose and 0.3% Phytagel was used for induction medium. Each treat- ment contained fi ve replicates.
Eco-type Genotype Growth regulator
2,4-D (2 mg l-1) Dicamba (4 mg l-1) NAA (2 mg l-1)
Spring Florida dwarf 66.74 79.17 51.79
Vågones vårrug 58.66 67.17 52.35
Auvinen 52.32 67.26 45.19
Rogo 52.17 56.23 47.09
Average 57.47B 67.46A 49.11C
Winter Zulpan 66.46 71.25 58.06
Amilo 62.85 66.15 50.00
Jussi 61.99 70.49 52.38
Anna 63.70 72.28 57.23
Riihi 48.40 66.36 45.91
Vågones 54.33 61.46 48.39
Wheeler 56.48 60.39 51.94
Voima 52.18 56.52 47.22
Average 58.30B 65.61A 51.39C
Total average 58.02B 66.23A 50.63C
LSD0.05 = 7.78 between auxins within spring genotypes LSD0.05 = 5.45 between auxins within winter genotypes LSD0.05 = 4.42 between auxins
2,4-D: 2,4-dichlorophenoxyacetic acid Dicamba: 3,6-dichloro-2-metoxy benzoic acid NAA: naphthaleneacetic acid
Table 4. Effects of ecotypes, genotypes, sugars and gel agents on somatic embryogenesis (embryogenic calli per 100 em- bryos) in rye embryo culture. AA medium with 4 mg l-1 Dicamba and 0.3% Phytagel was used for induction medium. Each treatment contained fi ve replicates.
Eco-type Genotype Sugar Gel agent
Sucrose (3%) Maltose (3%) Glucose (3%) Phytagel Agar
Spring ME80083 64.06 57.83 53.95 63.85 47.50
Florida dwarf 68.42 58.33 62.26 64.59 59.85
Vågones vårrug 61.74 58.97 56.13 61.00 57.42
Auvinen 56.48 48.74 41.21 61.38 55.21
OD 57.86 48.33 45.94 58.57 51.25
Gansu 61.95 52.70 48.11 58.44 51.85
Average 61.75A 54.15B 51.27B 61.31a 53.85b
Winter Zulpan 73.45 61.65 59.93 70.86 64.44
Amilo 65.87 60.7 49.49 67.39 52.63
Jussi 64.56 56.25 48.14 62.53 54.92
Anna 56.95 47.67 44.33 57.56 53.91
Riihi 54.93 49.24 43.78 54.14 39.72
Vagones 53.89 47.31 50.00 56.25 38.46
Wheeler 58.92 53.71 50.60 57.73 45.87
Voima 52.47 48.43 46.54 51.80 47.74
Average 60.13A 53.12B 49.10B 59.78a 49.71b
Total average 60.83A 53.56B 50.03B 60.44a 51.48b
LSD0.05 = 5.75 between sugars within spring genotypes LSD0.05 = 5.37 between sugars within winter genotypes LSD0.05 = 3.85 between sugars
LSD0.05 = 5.92 between gel agents within spring genotypes LSD0.05 = 5.18 between gel agents within winter genotypes LSD0.05 = 3.87 between gel agents
maltose and glucose. No signifi cant genotype and sucrose interactions were detected. Somatic em- broygenesis was signifi cantly improved by using the gel agent Phytagel (0.3%) (P < 0.001) com- pared to agar (0.7%) (Table 4).
Sugar is an essential requirement for all cul- ture media, providing carbon, energy and osmotic regulation. Sugar infl uences callus induction and green plant regeneration. In cereal crop somatic tissue culture, sucrose is most frequently used in culture media for somatic embryogenesis. Maltose has been shown to be the most effective sugar for increasing androgenesis and green plant regenera- tion in rye (Flehinghaus et al. 1991), wheat (Moieni 1997) and barley (Kuhlmann and Foroughi-Wehr 1989, However, our results revealed maltose (3%) to be an inferior carbohydrate source in compari-
son with sucrose (3%) for somatic embryogenesis of rye.
Infl uences of gelling agent in plant tissue cul- ture have been reported in previous studies (Koh- lenbach and Wernicke 1978, Lührs and Lörz 1987, Flehinghaus et al. 1991). Agar was widely used for culture medium solidifi cation. However, since Kohlenbach and Wernicke (1978) found the in- hibitory effects of agar, alternative gelling agents, such as agarose and gelrite, have been employed to replace agar. Agarose was superior to agar for in- creasing the effi ciencies of somatic embryogenesis in rye (Zimny and Lörz 1989) and barley (Lührs and Lörz 1987) embryo culture. Gelrite was bet- ter than agrose for embryogenic induction and regeneration rates in rye anther culture (Flehing- haus et al. 1991) and embryo culture (Popelka and
Table 5. Effects of ecotypes, genotypes and cold pre-treatment on somatic embryogenesis (embryogenic calli per 100 em- bryos) in immature embryo culture of rye. AA medium with 3% sucrose, 4 mg l-1 Dicamba and 0.3% Phytagel was used for induction medium. Each treatment contained fi ve replicates.
Eco-type Genotype Cold pre-treatment (weeks)
0 1 2 3 4 5 Spring Florida dwarf 63.69 73.81 71.43 67.78 62.00 53.47
Vagones vårrug 59.91 65.71 63.33 51.22 48.35 36.92
Auvinen 60.44 63.78 60.03 43.76 39.55 34.87
Gansu 64.29 65.69 66.67 52.75 50.77 35.70
Average 62.08A 67.25A 65.37A 53.88B 50.17B 40.24C
Winter Zulpan 64.33 73.84 72.22 63.64 62.71 56.82
Amilo 64.86 68.09 69.35 58.31 55.20 52.27
Jussi 53.51 74.04 68.02 48.74 38.41 41.23
Anna 56.57 69.01 63.70 54.59 53.60 49.60
Riihi 53.03 65.04 68.45 51.73 37.80 25.00
Vågones 54.81 58.26 54.45 50.00 49.66 48.21
Wheeler 57.58 60.93 60.66 56.88 53.79 48.94
Voima 45.85 51.66 58.33 48.75 48.48 46.03
Average 56.32B 65.11A 64.40A 54.08BC 49.96CD 46.01D Total average 58.24B 65.82A 64.72A 54.01BC 50.03C 44.09D LSD0.05 = 5.69 between cold treatments within spring genotypes
LSD0.05 = 5.38 between cold treatments within winter genotypes LSD0.05 = 4.25 between cold treatments
Altpeter 2001). Our results confi rmed the superi- ority of Phytagel (Gelrite) over agar for somatic embryogenesis in the embryo culture of rye.
Cold pre-treatment on somatic embryo- genesis and plant regeneration
Cold pre-treatment of immature embryo at 4ºC in darkness for 1–2 weeks signifi cantly improved somatic embryogenesis (Table 5) and plant regen- eration (Table 6). Duration of cold pre-treatment longer than three weeks reduced the culture effi - ciencies. No interactions of genotype × cold treat- ment on embryogenesis and plant regeneration were found.
In cereals cold pre-treatment of anthers is among the key factors infl uencing androgenesis and commonly employed to enhance the frequen- cy of embryogenesis and green plant regeneration (Thomas et al. 1975, Guo and Pulli 2000). Cold
pre-treatment of immature embryo was found to be favourable to somatic embryogenesis and plant regeneration in wheat (Maes et al. 1996) and triticale (Immonen 1996). A three-week cold pre-treatment of immature embryo at 4ºC before culture for wheat somatic embryogenesis and plant regeneration, and a two-week cold pre-treatment of immature embryos at 8ºC in culture for triticale plant regeneration were optimal. According to the results of our studies, one to two weeks of cold pre-treatment was benefi cial for both the embryo- genesis and plant regeneration of rye.
Differences of somatic embryogenesis between plant organs of rye
Somatic embryogenesis of different plant organs (embryo, infl orescence and leaf segment) of rye was tested (Table 7). Culture effi ciencies (embryo- genic calli per explant) for both spring and winter
Table 6. Effects of ecotypes, genotypes and cold pre-treatment on green plant regeneration (green plants per 100 calli) in rye embryo culture. 190-2 medium with 3% (w/v) sucrose, 3 mg l-1 BA and 0.45% Phytagel was used for regeneration medium. Each treatment contained fi ve replicates.
Eco-type Genotype Cold pre-treatment (weeks)
0 1 2 3 4 5
Spring ME8008 51.22 67.22 73.96 60.48 52.63 49.50
Florida dwarf 65.00 73.96 69.09 67.78 67.95 60.45
Auvinen 46.00 58.96 59.78 42.31 40.13 33.00
Average 54.07B 66.71A 67.61A 56.86B 53.57B 47.65B
Winter Zulpan 67.32 76.50 83.13 71.25 62.71 60.70
Amilo 68.79 76.67 70.34 66.81 65.00 62.37
Jussi 48.69 64.62 68.29 50.37 48.49 44.19
Anna 59.38 66.67 60.19 55.0 46.67 44.13
Riihi 43.39 52.11 57.85 47.59 43.69 34.80
Vågones 57.24 61.67 66.86 52.86 50.00 50.00
Wheeler 57.35 60.25 56.34 50.00 43.93 42.17
Voima 48.50 53.70 63.64 52.44 50.60 49.66
Average 56.33B 64.02A 65.83A 55.79B 51.39BC 48.50C Total average 55.72B 64.76A 66.32A 56.08B 51.98BC 48.27C LSD0.05 = 9.80 between cold treatments within spring genotypes
LSD0.05 = 5.85 between cold treatments within winter genotypes LSD0.05 = 5.01 between cold treatments
genotypes were considerably higher by embryo culture than by infl orescence culture, which were signifi cantly higher than by leaf tissue culture (P <
0.01). Callus induction frequencies of winter gen- otypes exceeded those of spring genotypes, but not signifi cantly. Frequencies for embryogenesis were 55.3% in embryo culture, 37.3% in infl orescence culture and 5.0% in leaf tissue culture. Signifi cant genotype × explant interaction in embryogenesis was found (P < 0.01).
In cereal crops immature embryo, immature infl orescence and leaf tissue have been commonly used as primary explant in the tissue culture of wheat (Ozias-Atkins and Vasil 1982, Gonzalez et al. 2001), rice (Jyoti and William 1996) and barley (Thomas and Scott 1985, Lührs and Lörz 1987, Timothy et al. 1993, Chang et al. 2003). In rye species, the somatic embryogenesis and plant regeneration from different explant sources of im- mature embryo (Lu et al. 1984, Zimny and Lörz 1989, Popelka and Altpeter 2001), immature infl o- rescence (Linacero and Vazquez 1990, Rakoczy- Trojanowska and Malepszy 1993) and leaf tissue
culture have been investigated. Immature embryo culture gave the best result, although other factors may have infl uenced the results. In our study, the highest embryogenesis frequency was from imma- ture embryo culture, being in agreement with pre- vious studies. The lowest frequency was from leaf tissue culture, which was much lower than that reported by Linacero and Vazquez (1986). This re- sult is likely explained by the different genotypes used in the experiments. In this study, interaction of genotype × explant played an important role in the somatic embryogenesis of rye. A similar inter- action has been found in barley (Ruiz et al. 1992).
Genotypic effects in embryo culture of rye
Table 8 shows that genotype signifi cantly infl u- enced somatic embryogenesis (P < 0.05) and green plant regeneration (P < 0.001). No signifi cant dif- ferences for embryogenesis and regeneration fre-
Table 7. Differences between plant organs in rye somatic embryogenesis. AA medium with 3% sucrose, 4 mg l-1 Dicamba and 0.3% Phytagel was used for induction medium. Each treatment contained fi ve replicates.
Eco-type Genotype Embryogenic calli per explants (%) Embryo Infl orescence Leaf segment
Spring ME80083 67.3 36.4 3.0
Florida dwarf 66.6 41.2 2.4
Vågones vårrug 57.8 43.3 2.1
Gansu 54.1 40.0 3.3
Auvinen 53.7 33.4 5.8
OD 51.1 27.2 4.0
Rogo 50.9 32.5 6.2
Kalhek K131 47.5 31.7 5.6
Floride 401 35.6 33.6 5.4
Average 53.84A 35.48B 4.20C
Winter Zulpan 73.2 44.7 3.9
Amilo 63.5 39.5 7.7
Jussi 59.4 43.7 6.3
Anna 60.4 41.5 4.5
Riihi 45.4 44.1 4.5
Vågones 51.7 39.7 6.4
Wheeler 50.6 25.7 10
Voima 51.2 36.4 4.6
Average 56.93A 39.41B 5.99C
Total average 55.29A 37.33B 5.00C
LSD0.05 = 6.23 between plant organs of spring genotypes LSD0.05 = 6.65 between plant organs of winter genotypes LSD0.05 = 4.41 between plant organs
quencies were found between winter and spring genotype samples. Frequencies of embryogenesis and regeneration varied from 43.30% (Lanzhou) to 70.53 % (Zulpan), and 34.29% (EM-1) to 71.08%
(Zulpan), respectively. A signifi cant (P < 0.01) cor- relation between embryogenesis frequency and re- generation frequency (r = 0.534) was found.
Genotype is an important infl uencing fac- tor in the plant tissue culture of cereal crops. In most species of cereals, genetic transformation is strongly dependent on genotype and effi ciency is largely determined by culture ability of the tissue (Maddock et al. 1983, Fennel et al. 1996). In the anther and microspore culture of rice (Gosal et al.
1997), wheat (Hu 1997) and rye (Guo and Pulli 2000), culture abilities are genetically controlled and culture effi ciencies are genotype dependent. In the immature embryo and infl orescence culture of wheat (Felföldi and Purnhauser 1992, Machii et al.
1998, Gonzalez et al. 2001), rice (Jyoti and Wil- liam 1996) and barley (Lührs and Lörz 1987). The genotypic effects on somatic embryogenesis and plant regeneration have also been noticed. For bar- ley, somatic embryogenesis and plant regeneration are two independent processes, and are controlled by independent genetic systems (Komatsuda et al.
1989, Stirn et al. 1995). In hexaploid wheat, the day length-sensitive allele ppd1 play major role in so- matic embryogenesis and plant regeneration (Ben et al. 1992). Rye is a crossing-pollinating species, and has a great variability due to its allogamous character. Even plants from the same genotype were not exactly genetically identical, but plants from same genotype are genetically more homo- geneous than plants from different genotypes. Dif- ferent genotypes could show different responses in tissue culture (Linacero and Vazquez 1990).
Rakoczy-Trojanowska and Malepszy (1995) sug-
Table 8. Response of genotypes to embryogenesis and plant regeneration in immature embryo culture of rye. AA medium with 3% sucrose, 4 mg l-1 Dicamba and 0.3% Phytagel was used for induction medium. 190-2 medium with 3% (w/v) sucrose, 3 mg l-1 BA and 0.45% Phytagel was used for regeneration medium. Each treatment contained fi ve replicates.
Eco-type Genotype No. of immature embryos
Embryogenic calli (% of embryos)
Green plants (% of embryogenic calli)
Spring ME-80083 69 66.42 60.14
KVL-7002 73 63.50 44.32
Florida dwarf 75 61.00 65.57
Vågones vårrug 72 60.96 50.00
Auvinen 65 55.10 51.55
OD 72 52.53 66.77
Gansu 69 54.40 56.82
Rogo 59 54.14 58.62
Kalhek K131 69 49.00 41.30
Florida 401 61 47.27 50.40
Average 68.4 56.43 54.55
Winter Zulpan 72 70.53 71.08
Amilo 70 66.90 70.94
Jussi 70 64.60 56.11
Bonel 74 59.90 48.17
Anna 70 58.10 57.14
Riihi 65 57.85 50.00
EM-1 61 56.50 34.29
Elvi 70 55.50 41.50
Vågones 54 54.59 59.89
Wheeler 71 53.90 54.30
Akusti 71 53.33 56.50
Voima 61 50.60 55.89
Danko 68 50.00 54.70
Sangaste 52 47.19 40.77
Vambo 61 45.27 42.80
Bylina 66 43.75 38.90
Lanzhou 68 43.30 48.20
Average 66.1 54.81 51.83
Total average 66.96 55.42 52.84
LSD0.05 = 14.93 between spring genotypes for embryogenic callus induction LSD0.05 = 14.96 between spring genotypes for green plant regeneration LSD0.05 = 15.21 between winter genotypes for embryogenic callus induction LSD0.05 = 14.95 between winter genotypes for green plant regeneration LSD0.05 = 14.92 between genotypes for embryogenic callus induction LSD0.05 = 14.77 between genotypes for green plant regeneration gested that in vitro response of rye seems to be a complex trait controlled by many genes co-operat- ing in different ways, and the regeneration ability were determined by recessive genes.
In the present study on the embryo culture of rye, both embryogenesis and regeneration differed signifi cantly among genotypes, thus proving the importance of genotype. Genotypic effects also have been observed in infl orescence culture of
rye (Linacero and Vazquez 1990, Rakoczy-Tro- janowska and Malepszy 1993). A correlation be- tween embryogenesis and regeneration for all gen- otypes tested in the present study was observed.
The genotypes with high embryogenesis also showed comparatively high regeneration. There was no difference in embryogenesis and regenera- tion between winter and spring genotype groups.
Similar results were also demonstrated in embryo,
infl orescence and scutellum culture of wheat and barely (Felföldi and Purnhauser 1992, Fransisco et al. 1999).
Effects of developmental stage of imma- ture embryos on somatic embryogenesis
Infl uence of embryo developmental stages (stage 1–6 of Zimny and Lörz 1989, size ranging from under 0.5 mm to 3 mm) were tested in this study.
Embryo developmental stages signifi cantly (P <
0.001) infl uenced the embryogenic induction (Ta- ble 9). Signifi cant highest embryogenesis (64.29%) was obtained from embryo sizes between 0.5–1 mm (about stage 3 of Zimny and Lörz 1989, Table 9).
Embryo size, which is an indicator for the de- velopmental embryonic stage, was a major infl u- encing factor in previous studies on the somatic embryogenesis of wheat, barley and rye. Previous studies have suggested optimal embryo sizes of 1.0–1.5 mm (Redway 1990), 0.5–2 mm (Özgen et al. 1996) and 1.7–1.9 mm (Maes et al. 1996) in wheat, 0.7–1.4 mm (Lührs and Lörz 1987), 1.1–
1.5 mm (Ruiz et al. 1992), 1.5–2 mm (Timothy et al. 1993) and 0.5–1.5 mm (Chang et al. 2003) in barley, 1–2 mm (Krumbiegel-Schroeren et al.
1984) in rye. Our results showed an embryo size of 0.5–1 mm (about stage 3 of Zimny and Lörz 1989) to be optimal for somatic embryogenesis. Small- er embryos (size < 0.5 mm, stage 1–2 of Zimny and Lörz 1989) responded to callus induction at a lower rate, while larger embryos (> 2mm, stages 5–6 of Zimny and Lörz 1989) germinated at faster
Table 9. Effects of ecotypes, genotypes and embryo size on somatic embryogenesis (embryogenic calli per 100 embryos) in rye embryo culture. AA medium with 3% sucrose, 4 mg l-1 Dicamba and 0.3% Phytagel was used for induction medium.
Each treatment contained fi ve replicates.
Eco-type Genotype Embryo Size (1–6 stages of Zimny and Lörz 1989)
< 0.5 mm Stage 1–2
0.5–1 mm Stage 3
1–2 mm Stage 4
2–3 mm Stage 5–6
Spring ME80083 43.33 68.75 59.74 47.37
Florida dwarf 56.25 68.39 61.86 54.40
Vågones vårrug 54.86 69.44 66.09 42.74
Auvinen 57.86 67.14 55.68 44.23
OD 43.16 56.30 48.83 44.58
Gansu 47.06 57.72 51.11 49.43
Rogo 44.85 59.18 53.70 51.06
Average 49.62C 63.85A 56.72B 47.69C
Winter Zulpan 64.15 77.94 75.92 58.46
Amilo 61.54 78.86 71.03 53.23
Jussi 44.17 64.46 58.76 51.25
Bonel 56.93 60.75 61.47 57.01
Anna 63.16 71.34 64.74 48.91
Riihi 50.63 54.13 53.61 41.27
Vagones 46.67 56.94 50.26 46.51
Wheeler 46.83 61.46 55.90 52.46
Voima 50.41 55.92 53.08 46.87
Average 53.83B 64.64A 60.53A 50.66B
Total average 51.99C 64.29A 58.86B 49.36C
LSD0.05 = 5.74 between embryo size within spring genotypes LSD0.05 = 5.35 between embryo size within winter genotypes LSD0.05 = 3.90 between embryo size
Table 10. Infl uence of subculture age on green plant regeneration (green plants per 100 calli) in rye embryo culture. AA medium with 3% sucrose, 2 mg l-1 2,4-D and 0.3% Phytagel was used as culture medium for embryogenic callus mainte- nance. Each treatment contained fi ve replicates.
Genotype Culture age (month)
1 2 3 4 5 6 7 8 9
Auvinen (sp) 53.7 50.4 44.1 37.6 34.6 27.3 16.4 11.5 6.8
Zulpan 74.3 68.8 59.3 50.3 46.5 38.6 32.5 26.3 19.4
Amilo 65.4 57.5 52.6 42.5 39.5 29.5 14.6 9.7 2.6
Jussi 56.8 53.7 49.2 41.4 33.6 24.4 11.3 7.6 1.7
Riihi 53.5 49.4 40.3 35.4 30.3 22.3 13.2 8.7 7.7
Vågones 62.4 59.4 51.5 45.3 38.7 33.3 22.7 18.4 11.2
Wheeler 56.7 51.9 41.4 34.2 28.2 23.6 19.4 13.3 5.8
Voima 58.1 50.3 45.4 38.4 33.0 23.5 18.9 12.6 8.4
Average 60.1 55.2 48.0 40.6 35.6 27.8 18.6 13.5 7.9
LSD0.05 = 4.34 between culture ages sp: spring genotype
rate and a higher percentage. These results were in agreement with of Krumbiegel-Schroeren et al.
(1984), Zimny and Lörz (1989) in the embryo cul- ture of rye.
Maintenance of morphological competence
Embryogenic calli were subcultured and green plant regeneration abilities were tested at each subculture. Regeneration ability signifi cantly (P <
0.001) decreased with increasing number of sub- cultures. With the exception of ‘Zulpan’ (19.4%) and ‘Vågones’ (11.2%), regeneration abilities of all genotypes were under 10% after 8 months of subculture (Table 10).
In cereal crops, embryogenic calli are suitable target tissues for genetic transformation, also be- ing suitable starting materials for cell suspension and protoplast cultures. Immature embryo derived calli have been frequently used for establishment of embryogenic cell suspension used as a source of totipotent protoplasts (Maddock 1987, Ahmed and Sagi 1993) and target tissue for genetic trans- formation (Becker et al. 1994, Dong et al. 2001).
Maintenance of embryogenic capability and re- generation potential has been a critical problem in an effi cient in vitro culture system (Lührs and
Lörz 1987, Chang et al. 2003). Prolonged dura- tion of subculture, has caused the gradual loss of embryogenic competence and regeneration potential (Bregitzer 1991, Jimenez and Bangerth 2001). Somatic embryogenesis and plant regenera- tion are genetically controlled, and the frequency of embryogenesis and regeneration is genotype dependent. Medium composition and physiologi- cal state of the donor plant affects the reaction of the explant under in vitro conditions (Lührs and Lörz 1987, Castillo 1998). Embryogenicity and regeneration ability has been maintained for 17 months in barley (Kachhwaha 1997), 36 months in wheat (Varshney 1996) and 34 months in rice (Utomo 1995). In our study, regeneration ability of embryogenic callus of rye was maintained for a maximum of 8 months for subculture. After 9 months subculture, regeneration ability was nearly lost. All green plants regenerated were normal in morphology and seed set.
Acknowledgements. We thank Mr. Jukka Karhu, Ms.
Mer vi Ylämäki, Ms. Marja Piirainen and Ms. Lioudmila Choumskaia for technical assistance, and the staff of the Laboratory of Plant Physiology and Molecular Biology for technical assistance. We thank Ms. Randi Kumpulai- nen for the English revision of the manuscript. This work was supported by the Finnish Ministry of Agriculture and Forestry.
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